Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, debilitating disease that is characterized by, among other things, abnormal hematopoiesis, complement-mediated intravascular hemolysis, and a propensity for thrombosis. See, e.g., Rosse and Nishimura (2003) Int J Hematol 77(2):121-124 and Brodsky (2008) Blood Rev 22(2):65-74. PNH is caused by a somatic mutation in the X-linked phosphatidylinositol glycan complementation class A (PIGA) gene, which encodes an enzyme that is necessary for the initial step of glycosylphosphatidylinositol (GPI) anchor biosynthesis. See Miyata et al. (1993) Science 259:1318-1320 and Bessler et al. (1994) EMBO J 13:110-117. GPI anchors attach a number of proteins to the surface of hematopoietic cells. These so called GPI-anchored proteins include, among others, complement regulatory proteins such as CD55 (DAF) and CD59. Depending on the type of mutation that befalls the PIGA gene, a partial or complete loss of GPI anchor biosynthesis can result, which corresponds to a partial or complete loss in the presence of GPI-anchored proteins (e.g., GPI-anchored CD55 and CD59) on the cell surface. See Rosse (1997) Medicine 76:63-93. The partial or complete absence of complement regulatory proteins on the surface of red blood cells (RBCs) results in the heightened sensitivity of these cells for complement-mediated lysis and associated symptoms of PNH in afflicted patients. See Nicholson-Weller et al. (1983) Proc Natl Acad Sci USA 80:5066-5070 and Yamashina et al. (1990) N Engl J Med 323:1184-1189.
Traditionally, diagnosis of PNH and monitoring of PNH patients involved analysis of CD55 and CD59 expression on the surface of RBCs and granulocytes using flow cytometry. Sutherland et al. (2009) Am J Clin Pathol 132:564-572. More recently developed diagnostic methods for PNH have employed a recombinant, non-lytic form of the bacterial protein aerolysin, which binds to GPI-anchors on the surface of hematopoietic cells. See U.S. Pat. No. 6,593,095 issued to Buckley and Brodsky. Both traditional and new methods have allowed medical practitioners to classify RBCs or white blood cells from PNH patients into one of three groups: Type I cells having normal or nearly normal cell-surface expression of GPI-anchored proteins; PNH Type III cells, which have nil or completely absent cell-surface expression of GPI-anchored proteins; and PNH Type II cells having an intermediate level of cell-surface expression of GPI-anchored proteins. Brodsky et al. (2000) Am J Clin Pathol 114:459-466. The characterization of Type II cells among white blood cell lineages has not been performed due to the difficulty in distinguishing these cells from normal Type I white blood cells.